Metabolites associated with the inclusion of atlantic GRO® seaweed supplementation as a methane mitigation strategy in the diet of replacement beef heifers.

INTRODUCTION: Dietary seaweed supplementation reduces enteric methane emissions in cattle, but the metabolic mechanisms remain unclear. METHODS: This study evaluated plasma metabolomic responses to Atlantic GRO® brown seaweed supplementation at 0.0%, 0.5%, 1.0%, 2.0% of diet dry matter, and replacement beef heifers were classified into low, moderate, and high methane emission groups. Plasma metabolites were analyzed using direct flow injection/liquid chromatography-tandem mass spectrometry (DFI/LC-MS/MS), and a total of 453 plasma metabolites were quantified. RESULTS: Significant dose-dependent metabolic shifts were observed, particularly at the 1.0% and 2.0% supplementation levels (p < 0.05). Compared with the control, the 2.0% seaweed group showed alterations in valine, TG.18:0_38:6, asymmetric dimethylarginine (ADMA), C2 acylcarnitine, trimethylamine (TMA), and inosine (p < 0.05, AUC ≥ 0.75, VIP score >1, MDA > 0), mainly involving lipid and amino acid metabolism. Stronger effects occurred at higher inclusion levels (1.0% and 2.0%). Methane-emission groups also differed in metabolites associated with energy metabolism and microbial fermentation pathways, with urea and ethylmalonic acid (EMA) identified as potential biomarkers (p < 0.05, AUC ≥ 0.75, VIP score > 1, MDA > 0). Overall, 152 metabolites were significantly affected by the interaction between seaweed supplementation and methane-emission group (p < 0.05), indicating that the metabolic response depended on methane phenotype. Metabolite differences were greatest between moderate and high methane emitters, with interaction-related changes mainly involving triglycerides, phosphatidylcholines, and microbial compounds, reflecting changes in lipid metabolism, nitrogen homeostasis, and gut microbial activity. Pathway analysis revealed enrichment of arginine biosynthesis, purine metabolism, and branched-chain amino acid metabolism, particularly at higher supplementation levels. DISCUSSION: Overall, Atlantic GRO® seaweed induced dose-dependent metabolic changes associated with methane reduction and supports the potential use of plasma metabolites as non-invasive biomarkers of enteric methane emissions in cattle.